Science of the Total Environment 622–623 (2018) 1572–1580

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Science of the Total Environment

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The toxicity of ionic liquid 1-decylpyridinium bromide to the algae

Scenedesmus obliquus: Growth inhibition, phototoxicity, and
oxidative stress
Dingdong Liu, Huijun Liu ⁎, Shengtao Wang, Jiazheng Chen, Yilu Xia
School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• High-concentration [DPy]Br inhibited

growth; low concentrations had
hormetic effects.
• [DPy]Br increased cell membrane per-
meability and damaged PSII.
• ROS, SOD, and CAT were stimulated by
[DPy]Br in S. obliquus.
• ROS content increase is an important
toxicological mechanism of [DPy]Br in
S. obliquus.

a r t i c l e i n f o a b s t r a c t

Article history: Although ionic liquids (ILs) are unlikely to act as air contaminants, their high solubility and slow degradation
Received 1 September 2017 make them a potential threat to the aquatic environment. The IL 1-decylpyridinium bromide ([DPy]Br) is a com-
Received in revised form 3 October 2017 mon type of pyridine IL, which has varied applications such as in extraction, separation, and catalytic synthesis.
Accepted 4 October 2017
Herein, the toxicity of [DPy]Br to S. obliquus is determined. Growth was inhibited by high-concentration
Available online 18 October 2017
[DPy]Br, whereas it had a hormetic effect at low concentrations. The IC50–96 h was approximately 0.06 mg/L.
Editor: Jay Gan The cell membrane permeability of S. obliquus increased with [DPy]Br concentration, indicating that [DPy]Br
can cause damage to the algae cell structure. Chlorophyll content decreased at high [DPy]Br concentration; chlo-
Keywords: rophyll fluorescence parameters, such as the maximum effective quantum yield of PSII (Fv/Fm), potential activity
1-decylpyridinium bromide of PSII (Fv/F0), yield of the photochemical quantum [Y(II)], and the non-photochemical quenching coefficient
Scenedesmus obliquus (NPQ) were affected, suggesting that [DPy]Br can damage PSII. The ROS fluorescent images revealed that the mor-
Growth inhibition phology of cells changed gradually from fusiform to round. High ROS levels were observed with high concentra-
Chlorophyll fluorescence tions of [DPy]Br, indicating that [DPy]Br induced oxidative stress on S. obliquus. The SOD and CAT activities
Oxidative stress
increased when the concentration was lower than IC50, whereas they decreased when the concentration was
higher than IC50. The relative ROS content was significantly correlated with growth inhibition rate, cell mem-
brane permeability, chlorophyll content, and SOD and CAT activities. The increase of ROS content in algal cells
is an important toxicological mechanism of [DPy]Br to S. obliquus.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail address: lhj@zjgsu.edu.cn (H. Liu).

https://doi.org/10.1016/j.scitotenv.2017.10.021
0048-9697/© 2017 Elsevier B.V. All rights reserved.
 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580
1. Introduction
Ionic liquids (ILs) have been widely used in extraction, catalysis,
synthesis, and separation of chemicals, as well as in other fields
(Hallett and Welton, 2011). ILs have been gradually gaining popu-
larity as novel alternatives to traditional organic solvents because
of their good solubility, catalytic properties, non-flammability,
low vapor pressure, wide electrochemical window, and high stabil-
ity (Kárászová et al., 2014). However, their environmental friendly
characteristics have been questioned in recent years, especially in
the aquatic environment (Cvjetko et al., 2014). The properties of
stability and water solubility make it easier for ILs to accumulate
in water and trigger a variety of aquatic environmental problems.
Several studies found that ILs have great influence on aquatic ani-
mals (Zhang et al., 2017), aquatic plants (Deng et al., 2017a,
2017b), and aquatic microorganisms (Rantamäki et al., 2017),
thus indicating that more attention should be paid to the environ-
mental toxicity of ILs to aquatic organisms (Amde et al., 2015;
Egorova and Ananikov, 2014).
Pyridine ionic liquids are a common kind of ILs. Recently,
pyridine-containing anion-based ILs have been found to be highly ef-
ficient for catalyzing the cycloaddition reactions of atmospheric CO2
with epoxides at room temperature (Luo et al., 2014). With pyridine
ILs becoming widely used, the amount of pyridine salt exposure to
the environment is also getting higher. However, basic toxicology re-
search on pyridine salts is still limited. Therefore, the mechanism re-
sponsible for the toxicity of pyridine ILs needs more attention. 1-
decylpyridinium bromide ([DPy]Br), containing a long alkyl chain
and a single anionic group, is a common kind of pyridine ILs. The
ecotoxicity study of [DPy]Br is scarce, and no study is addressing
the toxicity of [DPy]Br to algae.
Photosynthesis is not only the sum of a series of complex chemi-
cal reactions, but also the basis for survival of plants. Photosynthesis
is one of the key physiological processes that maintain the normal
operation of cells when algae are under stress (Bubalo et al., 2014).
In recent years, monitoring photosynthesis of polluted plants using
chlorophyll fluorescence has become one of the most rapid and
non-invasive methods (Kumar et al., 2014). The main energy
absorbed by chlorophyll molecules is used for photochemical reac-
tions, and the excess is dissipation as heat or emitted as fluorescence
(Maxwell and Johnson, 2000). Chlorophyll fluorescence will be
quenched by various photochemical and non-photochemical
processes because of pollutants or their environmental effects
(Neil R. Baker, 2008).
When exposed to an adverse environment, one of the immedi-
ate responses of algae is to excessively produce reactive oxygen
species (ROS), such as hydrogen peroxide (H 2 O 2 ), singlet oxygen
( 1 O 2 ), the superoxide radical (O 2 • − ), and the hydroxyl radical
(OH• − ) (Apel and Hirt, 2004). Algae have a sophisticated system
that involves non-enzymic antioxidant molecules, such as glutathi-
one, ascorbate, flavonoids, α-tocopherol, and carotenoids, and ef-
fective antioxidant enzymes, such as ascorbate peroxidase (APX),
glutathione reductase (GR), glutathione S-transferase (GST), su-
peroxide dismutase (SOD), and catalase (CAT). This system can
self-regulate ROS levels and allow cells to adapt to the oxidative
stress (Qian et al., 2016).
In the present study, the growth inhibition of Scenedesmus
obliquus (S. obliquus) caused by 1-decylpyridinium bromide
([DPy]Br) was examined. The effects of [DPy]Br on cell membrane
permeability, chlorophyll content, and the photosynthetic system
of S. obliquus were measured. The ROS levels, the distribution
of ROS, and antioxidant enzyme activities (SOD and CAT) in
S. obliquus cells were measured to evaluate the oxidative stress
caused by [DPy]Br. Regression analysis between ROS levels and
other indexes were conducted, and the mechanism responsible for
the toxicity of [DPy]Br was revealed.
1573
2. Materials and methods
2.1. Algal culture
The microalgae S. obliquus was obtained from the Institute of Hydro-
biology, Chinese Academy of Sciences (Wuhan, China), and was cul-
tured in HB-4 medium, which was prepared according to the Chinese
National Environmental Protection Agency Guidelines 201 (CNEPA,
1990). The HB-4 medium was sterilized in an autoclave (Hirayama
HVE-50) before use. The composition of the HB-4 medium including
distilled water and the following chemical ingredients: (NH4)2SO4
200 mg/L; Ca(H2PO4)2·H2O + (CaSO4·H2O) 30 mg/L; MgSO4·7H2O
80 mg/L; NaHCO3 100 mg/L; KCl 23 mg/L; FeCl3 1.5 mg/L; and 0.5 mL
of soil leaching solution. The S. obliquus were cultured and tested
regularly in an environmental chamber with standard lighting and tem-
perature conditions (16:8 light: dark cycle; illumination 3000–4000 lx;
25 °C) (Liu et al., 2017). All culture flasks were shaken numerous times
per day.
2.2. Chemicals
The IL 1-decylpyridinium bromide ([DPy]Br) used for testing was
purchased from Chengjie Chemical Co. LTD (Shanghai, China), with pu-
rity is 99%. Fluorescein acetate (FDA) and H2DCFDA was purchased from
Sigma Aldrich. All other reagents were analytical grade.
2.3. The growth inhibition tests
The growth inhibition test was performed according to the OECD
Guideline 201 (OECD, 2006). Algae with log phase were inoculated
into a series of IL concentrations in sterilized flasks to reach a final den-
sity of 8.0 × 105 cells/mL in the final 100 mL solution. The concentration
of [DPy]Br was set as follows: 0, 0.001, 0.002, 0.005, 0.01, 0.03, 0.05, 0.06,
0.08, 0.1, 0.3, and 0.5 mg/L. The S. obliquus cell density was measured at
680 nm using a TU-1901 spectrophotometer (Wen et al., 2011) after 24,
48, 72, 96, 120, and 144 h exposure. The algae cell density was calculat-
ed according to the following linear equation: Y = 310.8X − 2.173,
where Y is the density of the average algae cells (× 105 cells/mL) and
X is the absorbance at 680 nm. The relative inhibitions (RI) of algal
growth by [DPy]Br were calculated, and the concentration of [DPy]Br
leading to a 50% inhibition (IC50) of algal growth was calculated using
a logistic model (Laguerre et al., 2009).
[DPy]Br concentrations for all the following tests were set as follows:
0, 0.001, 0.01, 0.03, 0.06, 0.08, and 0.3 mg/L. The algae samples were
collected after 96 h treatment.
2.4. Effect of [DPy]Br on cell membrane permeability
Algae cells absorb FDA though the passive absorption of the cell
membrane, and its nonspecific esterases hydrolyzed FDA to produce
the fluorescent molecule fluorescein. The test evaluated cell membrane
permeability by measuring the fluorescent molecule fluorescein.
Fresh FDA stock solutions were prepared daily in acetone and kept at
− 20 °C. After 96 h, algal cell samples (3 mL) were centrifuged at
6000 rpm for 10 min, and resuspended in fresh HB-4 medium to obtain
a final cellular density of 4.0 × 105 cells/mL. FDA solution was added to
obtain a final concentration of 2 μM. Hydrolysis was conducted in the
first 10 min, the initial hydrolysis rates for FDA were obtained from
the slope of the linear regression of fluorescence intensity versus time,
and the slope was measured using a fluorescence spectrophotometer
(F-4600, Hitachi, Japan) at an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. The initial hydrolysis rates for FDA rep-
resent the cell membrane permeability (Bernard et al., 2000).
1574 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580
2.5. Effects of [DPy]Br on photosynthesis
2.5.1. Chlorophyll concentration
After 96 h treatment, 40.0 mL algae were taken and centrifuged at
4000 rpm for 10 min. The supernatant was discarded and 90% acetone
solution was added to the cell pellet in a final volume of 10.0 mL. After
24 h extraction, the samples were measured according to the method
of Mackinney (1941) using the following equations:
CA ¼ 12:7 OD663 −2:69 OD645
CB ¼ 22:9 OD645 −4:680 D663
CT ¼ CA þ CB
Where CA, CB, and CT are the concentration of chlorophyll a (chl a), chlo-
rophyll b (chl b), and total chlorophyll (chl (a + b)), respectively.
2.5.2. Chlorophyll fluorescence test
After 96 h treatment, 80.0 mL algae had been concentrated to 1.0 mL
and placed in a 96-well plate and dark-adapted for 20 min. The chloro-
phyll fluorescence test was conducted using a pulse-amplitude-
modulated fluorometer (Maxi-version of the Imaging-PAM, Heinz
Walz GmbH, Germany). The minimum fluorescence (F0) and maximum
fluorescence (Fm) were measured, whereas the chlorophyll fluores-
cence parameters of photosynthetic system II (PSII), such as the variable
fluorescence (Fv), the maximum quantum yield of PSII photochemistry
(Fv/Fm), and its more sensitive form Fv/F0 were calculated as follows
(Lichtenthaler et al., 2005):
F v ¼ F m −F 0
F m −F 0
F v =F m ¼
Fm
F m −F 0
F v =F 0 ¼
F0
The rapid light response curves were then constructed by exposing
the algae to nine steps of increasing photosynthetically active radiation
(PAR, approximately 1, 35, 80, 145, 230, 335, 460, 610, and 800
μmol/m2·s) (Lefebvre et al., 2011). Each irradiance step was set to
20 s. The chlorophyll fluorescence parameter yield of photochemical
quantum [Y(II)] and non-photochemical quenching coefficient (NPQ)
were calculated by the following formulas (Kim et al., 2015; Liu et al.,
2015b):
0 1 mL of H2O2 (30 mmol/L). The total testing time was 3 min and it
F m −F t
Y ðIIÞ ¼ ΔF v =F m ¼ 0 ð7Þ
Fm
Fm
NPQ ¼ 0 −1
Fm
Where Fm′ represents the maximum fluorescence under actinic light
(μmol/(m2·s)), Ft is the real-time fluorescence (μmol/(m2·s)).
2.6. Oxidative stress of [DPy]Br
2.6.1. Reactive oxygen species (ROS) levels
The method for determining ROS level was modified from that of
Stefanie and Katja (2008). The H2DCFDA stock solution (10 mM) was
prepared in methanol and maintained at − 20 °C. The stock solution
was diluted 1000-fold in HB-4 medium before use. Algal cell samples
(3 mL) were centrifuged at 4000 rpm for 10 min and the supernatant
was discarded. H2DCFDA (0.3 mL, 10 μM) was added to the HB-4 medi-
um, for a final volume of 3 mL. Then, samples were incubated in a water
bath at 37 °C in the dark. After 30 min of incubation, the samples were
centrifuged at 4000 rpm for 10 min, washed twice, and resuspended
in the HB-4 medium. The H2DCFDA in algal cells was hydrolyzed to
H2DCF by cellular esterases. H2DCF is converted to DCF immediately
and then fluoresces in the presence of ROS (Wang et al., 2011). The
ROS levels were quantitatively determined using a fluorescence spec-
trophotometer, with excitation at 485 nm and emission at 530 nm.
Changes in ROS content compared to the control were evaluated ac-
cording to Hong et al. (2009):
ð1Þ Relative ROS sensor level ð%Þ
1 þ ðMean DCF fluo:½RMIM T‐Mean DCF fluo:½controlÞ
¼  100%ð9Þ
ð2Þ Mean DCF fluo:½control
ð3Þ where the relative ROS sensor level represented the percent increase in
ROS formation in response to RMIM T treatment as compared with that
of the control.
The distribution of ROS in S. obliquus cells was examined using Leica
TCS SP5 Laser scanning confocal microscopy (LSCM) with excitation at
485 nm and emission at 530 nm.
2.6.2. Enzyme activity
The S. obliquus cell samples (40 mL) were centrifuged at 4000 rpm
for 10 min. The resultant pellet was washed twice with phosphate buff-
er (50 mM, pH 7.8), and transferred to the buffer solution for a final vol-
ume of 5 mL. The cells were disrupted by an ultrasonic cell disrupter in
an ice bath using an Ultrasonic Cell Disruption System at 800 W, operat-
ing and resting for 3 s each. Total cell disruption time was 5 min. The
disrupted cells were then centrifuged at 10,000 g for 10 min at 4 °C.
The supernatant containing the enzymes was collected and stored at
−80 °C for future analysis.
ð4Þ
The total soluble protein level was measured using bovine serum al-
bumin (BSA) as the standard, according to Bradford (1976). SOD activity
ð5Þ was measured at 560 nm and calculated as the amount of enzyme re-
quired for 50% inhibition of NBT reduction. The SOD activity was calcu-
lated as follows:
ð6Þ
A0 −As
SODðU=mg ProteinÞ ¼ lim ð10Þ
0:5  A0  C protein  V x→∞
where A0 represents the absorbance of the blank, As represents the ab-
sorbance of the sample, Cprotein represents the concentration of protein
in the sample (mg/mL), and V represents the volume of the extracted
antioxidant enzyme used in the test.
Catalase activity (CAT) was detected at 240 nm using H2O2 as the
substrate. The reaction was initiated by adding 0.1 mL of extracted anti-
oxidant enzyme, 2.9 mL of phosphate buffer (0.05 mol/L, pH 7.8), and
was measured at 30 s, compared against the blank containing phos-
phate buffer (0.05 mol/L, pH 7.8). One unit of CAT activity was defined
as the amount of enzyme that decomposed H2O2 in 1 min at 240 nm
(Feierabend and Engel, 1986). The CAT activity was calculated as
ð8Þ
follows:
ΔA240
CATðU=mg ProteinÞ ¼ ð11Þ
t  C protein  V
where ΔA240 is the change in absorbance of the sample within 3 min and
t = 3 min.
2.7. Statistical analysis
Three replicates were tested for each treatment. Data were analyzed
using SPSS 19.0 and one-way analysis of variance (ANOVA) were per-
formed to compare mean values. Differences were compared using
Tukey multiple comparison tests followed by Dunnett test at a signifi-
cance level of 5%. All results are presented as the mean of three
replicates ± standard deviation (S.D.).
 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580
Fig. 1. Concentration-response data of S. obliquus treated by [DPy]Br for 24, 48, 72, 96, 120,
and 144 h and the concentration-response fitting curve using the logistic model. Note:
logistic model: y = A2 + (A1 − A2)/[1 + (x/x0)P], where y represents the relative
inhibition of algal growth (%), x represents the concentration of [DPy]Br (mg/L), A1 and
A2 are the upper and lower bounds for the function value y in the equation, x0
represents the IC50 (mg/L), and P is a constant.
3. Results and discussion
3.1. The growth inhibition of S. obliquus by [DPy]Br
Biological activity is modulated by interaction with water and even
by involvement of specific solvation effects (Egorova et al., 2017), the
[DPy]Br solution was distilled water with HB-4 medium for alga growth.
The growth inhibition rate of [DPy]Br treatments significantly increased
(p b 0.05) with increasing concentration given the same exposure time,
showing a dose-response relationship. The growth inhibition rate was
negative in low concentrations and exhibiting a hormetic effect. The
hormetic effect existed at all exposure times in 0.001 mg/L, the growth
inhibition rate at exposure times of 24, 48, 72, 96, 120, and 144 h was −
7.34%, − 3.12%, − 1.89%, − 1.11%, − 0.48%, and − 0.34%, respectively.
Similar hormetic effects of ILs were obtained in the research of
Nancharaiah and Francis (2015) and Zhu et al. (2016).
The growth inhibition rate increased at 48 h or 72 h and decreased
with exposure time at lower concentrations (b 0.06 mg/L), whereas in-
hibition increased with increasing time at higher concentrations
(N0.08 mg/L) (Supplementary material Table 1). The results suggest
that the effect of [DPy]Br on algal growth can be reversed with increas-
ing exposure time at low concentrations (lower than the IC50). Mecha-
nistically, a low concentration treatment over time may result in the
allocation of damage repair factors, or this compensation response
could be integrated into other signal pathways, which would lead to re-
duced damage or even enhanced resistance (Calabrese, 2015).
The concentration–response fitting curves of [DPy]Br using a logistic
model are shown in Fig. 1. The IC50 values calculated by the logistic
model and those obtained from the curves are shown in Table 1. The re-
sults showed the substantial difference between the 24 h-IC50 values.
This may have resulted because the maximum growth inhibition rate
of algae exposed to ILs for 24 h was only approximately 60%, which is
not suitable for a logistic model. The IC50 values of [DPy]Br decreased
Table 1
The IC50 values of [DPy]Br (mg/L).
Method 24 h-IC50 48 h-IC50 72 h-IC50
L 0.052 (0.047, 0.057) 0.050 (0.045, 0.056) 0.056 (0.050, 0.062)
D 0.149 0.055 0.060
Note: Method “L” means the IC50 value was obtained using the logistic model, method “D” means the IC50 value was determined from the curve. The value within brackets represent 95%
confidence intervals.
1575
Fig. 2. Cell membrane permeability of the S. obliquus membrane treated by [DPy]Br after
96 h. Note: The bars represent mean ± SD. The different letters indicate significant
differences at p b 0.05 according to Tukey's multiple range test.
from 24 to 48 h, then remained stable with increasing exposure time.
The IC50 values of [DPy]Br treatments were 0.149, 0.055, 0.060, 0.061,
0.062, and 0.063 mg/L, respectively. The same results were detected in
the study of Fu et al. (2015), which showed that the most sensitive pe-
riod in algal growth inhibition tests was at an exposure time of 48 h.
3.2. Effects of [DPy]Br on cell membrane permeability
The first self-protection barrier of algae is composed of a cell wall
and cell membrane. The cell membrane with its permselectivity plays
an important role in protecting the cell. The membrane permselectivity
changes after exposure to pollution. A number of studies have shown
that ILs can damage cell membranes of organisms, leading to functional
disorder in the cells, inhibiting normal development and growth (Gal
et al., 2012). Membrane permeability increased with [DPy]Br concentra-
tion exposure compared with that of the control (p b 0.05) (Fig. 2). Cel-
lular membrane permeability was 1.06, 1.56, 2.66, 5.21, 6.24, and 7.48
times that of the control group in 0.001, 0.01, 0,03, 0.06, 0.08, and
0.3 mg/L [DPy]Br treatment, respectively. High concentrations of
[DPy]Br caused severe damage to algae cells, and membrane permeabil-
ity remained at a high level.
3.3. Effects of [DPy]Br on chlorophyll concentration
The concentration of chl a, chl b, and chl (a + b) decreased with in-
creasing [DPy]Br concentration, except for the 0.001 mg/L treatment
(Table 2). This is in concurrence with the results of growth inhibition
tests and cell membrane permeability. Higher cell membrane perme-
ability in treatment with higher concentrations could allow the ILs to
enter the cell more easily, where they damaged the thylakoids in the
chloroplast, which blocked chlorophyll synthesis (Liu et al., 2015a).
The important part of photosynthesis is chlorophyll, including chl a,
which is considered the basis upon which to estimate photosynthetic
and respiratory rates, and chl b, which is a complementarity secondary
96 h-IC50 120 h-IC50 144 h-IC50
0.057 (0.051, 0.064) 0.060 (0.052, 0.067) 0.061 (0.056, 0.065)
0.061 0.062 0.063
1576 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580

Table 2
Effect of [DPy]Br on the chlorophyll concentration of S. obliquus after 96 h of exposure.

[DPy]Br (mg/L) Chl a Chl b T Chl Chl a/Chl b

0 1.341 ± 0.021a 0.459 ± 0.002a 1.801 ± 0.030a 2.926 ± 0.159b

0.001 1.384 ± 0.034a 0.455 ± 0.002a 1.839 ± 0.043a 3.049 ± 0.143c
0.01 1.195 ± 0.036b 0.330 ± 0.025b 1.525 ± 0.017b 3.642 ± 0.389e
0.03 0.872 ± 0.019c 0.228 ± 0.010c 1.100 ± 0.009c 3.823 ± 0.251f
0.06 0.531 ± 0.013d 0.154 ± 0.025d 0.685 ± 0.037d 3.510 ± 0.480d
0.08 0.395 ± 0.012e 0.093 ± 0.022e 0.487 ± 0.035e 4.391 ± 0.886 g
0.3 0.143 ± 0.075f 0.075 ± 0.019ef 0.219 ± 0.033f 2.011 ± 0.653a

Note: The values represent mean ± SD. The different letters indicate significant differences at p b 0.05 according to Tukey's multiple range test.

chlorophyll for chl a (Bubalo et al., 2014). The increase of growth inhibi-
tion may be directly caused by a breach in chlorophyll synthesis (Ashraf
and Harris, 2013).
3.4. Effects of [DPy]Br on chlorophyll fluorescence parameters
The composition of pigment systems, excitation energy transfer,
changes in the efficiency of photosynthesis, and heat dissipation can
be effectively reflected by chlorophyll fluorescence (Kumar et al.,
2014). Chlorophyll fluorescence was used to examine the polyaromatic
hydrocarbons (PAHs) effects on plants (Petrová et al., 2017) and heavy
metal toxicity on algae (Koppel et al., 2017).
3.4.1. Effects of [DPy]Br on F0, Fv/Fm, and Fv/F0
The images of the algae were recorded under various modes after
the sample was placed in an I-PAM chlorophyll fluorometer. The color
of algae images changed from blue to green and finally to orange with
the increasing concentration of [DPy]Br (Fig. 3A), showing a dose-
dependent relationship. When all antenna pigment complexes associat-
ed with the photosystem are assumed to be open (dark adapted), the F0
value is considered to be at minimal fluorescence (Liu et al., 2015b). The
F0 value increased with increasing [DPy]Br concentration (Table 3),
showing values that were 1.03, 1.09, 1.49, 2.51, and 3.79 times that of
the control, respectively. The reason of the F0 value increasing may be
the reversible or irreversible inactivation of PSII or damage of the thyla-
koid membrane, inhibiting the synthesis of photosynthesis (Liu et al.,
2015a).
The Fv/Fm values decreased with the addition of [DPy]Br. The Fv/Fm
values decreased from 0.672 to 0.136 and the Fv/F0 values decreased
from 2.045 to 0.158 with increasing [DPy]Br concentration. Fv/Fm is the
maximum quantum yield of PSII photochemistry, and lower Fv/F0
value occurred during stress conditions compared to that of the control.
This ratio has extraordinary abilities to indicate the photosynthetic per-
formance of plants (Lichtenthaler et al., 2005). The increase in [DPy]Br
concentration could result in an increased proportion of QB-non-
reducing PSII reaction centers, and an inhibition of the transfer of

Fig. 3. The effects of [DPy]Br on chlorophyll fluorescence parameters after 96 h. A. The Fv/Fm images of 96 wells filled with suspensions of S. obliquus. Note: the color scale is shown at the
bottom. The values of Fv/Fm decreased from left to right with the addition of [DPy]Br, and the color changed from blue to orange. B. Change in Y(II); C. Change in NPQ.
 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580 1577

Table 3
Effect of [DPy]Br on F0, Fv/Fm, and Fv/F0 after 96 h of exposure.

[DPy]Br (mg/L) F0 Fm Fv Fv/Fm Fv/F0

0 0.075 ± 0.001a 0.228 ± 0.002ab 0.153 ± 0.002a 0.672 ± 0.006a 2.045 ± 0.051a
0.001 0.077 ± 0.001a 0.236 ± 0.002b 0.159 ± 0.003ab 0.671 ± 0.006a 2.043 ± 0.053a
0.01 0.082 ± 0.001a 0.246 ± 0.004c 0.164 ± 0.005b 0.668 ± 0.007a 2.012 ± 0.065a
0.03 0.112 ± 0.002b 0.220 ± 0.004a 0.108 ± 0.003c 0.491 ± 0.009b 0.964 ± 0.036b
0.06 0.188 ± 0.004c 0.267 ± 0.004d 0.081 ± 0.004d 0.300 ± 0.012c 0.429 ± 0.025c
0.08 0.284 ± 0.006d 0.328 ± 0.003e 0.045 ± 0.004e 0.136 ± 0.011d 0.158 ± 0.015d

Note: The values represent mean ± SD. The different letters indicate significant differences at p b 0.05 according to Tukey's multiple range test.

excitation energy from the phycobilisome to the PSII reaction centers
(Kumar et al., 2014).
3.4.2. Effects of [DPy]Br on rapid light response curves
The Y(II) is the PSII photochemical efficiency in light-adapted plants,
which reveals the physiological state (Kumar et al., 2014). Y(II) de-
creased with increasing [DPy]Br concentration (Fig. 3B). At 145 μmol/
(m2·s) of PAR, the Y(II) was 95.8%, 59.8%, 20.3%, and 17.2% of that of
the control in 0.01, 0.03, 0.06, and 0.08 mg/L [DPy]Br treatments, respec-
tively, which indicates that [DPy]Br may affect carbon fixation and as-
similation in algal cells, which further impedes the formation of
assimilatory power (such as the production of NADPH and ATP) (Liu
et al., 2015b). The [DPy]Br can damage PSII, and the degree of damage
increases with increasing concentration of [DPy]Br, which affects the
normal photosynthesis of algae and causes irreversible damage.
NPQ refers to the non-chemical quenching of fluorescence caused by
heat dissipation, and it is also an important protective mechanism for
PSII (Kumar et al., 2014). NPQ is generally used as an indicator of the ef-
fect of a factor on the inhibition of algae photosynthesis. Studies have
shown that damage to chloroplasts by poisons may lead to a reduction
in NPQ values (Schreiber et al., 2007). NPQ decreased with increasing
concentration of [DPy]Br (Fig. 3C). At 145 μmol/(m2·s) of PAR, the

Fig. 4. ROS level and ROS production in S. obliquus exposed to [DPy]Br after 96 h. Data that are significantly different (p b 0.05) are denoted with different letters. Fluorescent images were
collected by LSCM.
1578 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580

Fig. 5. Effect of [DPy]Br on SOD and CAT activity in S. obliquus after 96 h. Note: The bars represent mean ± SD. The different letters indicate significant differences at p b 0.05 according to
Tukey's multiple range test.

Fig. 6. Regression analysis between ROS level with algae growth inhibition, cellular membrane permeability, enzyme activity, and chlorophyll concentration.
 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580
NPQ was 89.2%, 55.2%, 26.3%, and 20.5% that of the control in 0.01, 0.03,
0.06, and 0.08 mg/L [DPy]Br treatments, respectively, and that of 335
μmol/(m2·s) of PAR was 90.1%, 54,6%, 26.4%, and 24.9%, respectively.
NPQ was used as a measure of structural damage to the photosynthetic
apparatus, specifically reduced NPQ represents a modification of acces-
sory pigments (Corcoll et al., 2012). Y(II) and NPQ decreased with in-
creasing [DPy]Br treatment, which indicated that [DPy]Br has a
substantial effect on chloroplasts in algal cells, which affects the normal
utilization of light energy in PSII.
3.5. Effect of [DPy]Br on oxidative stress
3.5.1. Effect of [DPy]Br on ROS distribution and ROS levels
Reactive oxygen species (ROS) include superoxide radicals (O2•−),
hydrogen peroxide (H2O2), hydroxyl radicals (OH•−), and peroxide rad-
icals (RCOO•). All are highly reactive oxidants. ROS alter redox-
mediated cellular processes and stress the cell, which damages cell
membranes and other cellular components, resulting in a variety of dif-
ferent cellular responses in the plant, including programmed cell death
(PCD), development changes, and hormone signaling (Saleh et al.,
2016).
The effects of [DPy]Br on ROS generation in S. obliquus cells are
shown in Fig. 4. The ROS level increased significantly with increasing
[DPy]Br concentration, it was 115.03%, 131.79%, 173.33%, 198.25%,
235.50%, and 270.73% that of the control, respectively in 0.001, 0.01,
0.03, 0.06, 0.08, and 0.3 mg/L [DPy]Br treatment. The results showed
that there was a significant positive correlation between the intracellu-
lar ROS level and the [DPy]Br concentration. This result is consistent
with many previous studies regarding ILs. Increased ROS production
in marine diatoms (Deng et al., 2017a, 2017b) was also observed with
IL exposure.
The amount of green fluorescence was clearly increased in the treat-
ments, especially at high concentration. Brighter green fluorescence
was observed in higher concentrations of [DPy]Br (Fig. 4). The result
suggested that [DPy]Br stimulated ROS production and the ROS genera-
tion was concentration-dependent for [DPy]Br in S. obliquus. As the con-
centration increases, the morphology of the cells gradually changed
from fusiform to round, which demonstrated that the accumulation of
ROS placed stress on algal cells and changed the normal morphology
of the cells.
3.5.2. Effect of [DPy]Br on antioxidase
When S. obliquus was under stress because of the pollutant, it acti-
vated defense mechanisms to maintain ROS at an affordable level. The
antioxidant enzyme system secreted SOD to catalyze the superoxide
radical (O− 2 ) and produce hydrogen peroxide (H2O2), then it secreted
CAT to decompose H2O2 into non-toxic H2O and O2. Excessive ROS
was removed by this mechanism (Khare et al., 2015). The SOD and
CAT activities could be used as an indicator of cellar antioxidant levels.
The results showed that the SOD and CAT activities in the treatment
groups were significantly higher than those in the control group (p b
0.05), which suggested that [DPy]Br caused increased oxidative stress
and antioxidant enzyme activities (Fig. 5). The SOD activities were
1.08, 1.36, 1.58, 1.42, 1.21, and 1.07 times that of the control, respective-
ly, in 0.001, 0.01, 0.03, 0.06, 0.08, and 0.3 mg/L [DPy]Br treatment. Sim-
ilarly, the CAT activities first increased as [DPy]Br concentration rose,
but then decreased at high [DPy]Br concentrations. These results sug-
gested that the SOD and CAT activities increased as the [DPy]Br concen-
tration increased and decreased when the concentration was higher
than the IC50 value.
3.5.3. Regression analysis between relative ROS level and other indexes
Linear regression was used to model the relationship between rela-
tive ROS levels and growth inhibition rate, cell membrane permeability,
chlorophyll concentration, and enzyme activity (SOD and CAT) (Fig. 6).
The coefficient of determination (R2) for growth inhibition rate and
1579
relative ROS level was 0.9475, which indicated that the regression line
perfectly fits the data. R2 for cell membrane permeability and relative
ROS level was 0.9571, indicating that the relative ROS levels were line-
arly correlated with cell membrane permeability. It was shown that
the disruption of the cell membrane was caused by lipid peroxidation
of ROS. Free radicals denatured the proteins and lipids in the cell mem-
brane, thereby altering cell membrane fluidity and ion transport, lead-
ing to a change in cell membrane permeability (Pacurari et al., 2012).
The same result was also obtained from the linear regression between
relative ROS level and chlorophyll concentration. The R2 for ascending
enzyme activity and relative ROS level was 0.9104 (SOD) and 0.9528
(CAT), and that of the descending phase was 0.9600 (SOD) and 0.9596
(CAT). The linear regression relationship between relative ROS level
and enzyme activity showed that the accumulation of ROS controlled
the effects on enzyme activity.
[DPy]Br caused oxidative stress in S. obliquus and induced excessive
ROS production, which led to increases in cell membrane permeability,
caused damage to PSII in algae, destroyed the normal operation of the
enzyme system, and finally inhibited growth. The increase in ROS con-
tent in algal cells is an important toxic mechanism of [DPy]Br on
S. obliquus.
4. Conclusions
The present study demonstrated the effect of [DPy]Br on algae.
Growth was inhibited by [DPy]Br in high concentrations, whereas it
had a hormetic effect at low concentrations. Cell membrane permeabil-
ity increased with [DPy]Br concentration, whereas chlorophyll content
decreased. The chlorophyll fluorescence parameters (F0, Fv/Fm, Fv/F0,
Y(II), and NPQ) were affected by [DPy]Br, indicating that the high
[DPy]Br concentration would damage PSII. The transfer of excitation en-
ergy was inhibited, and photosynthetic efficiency was reduced. The ROS
level increased in [DPy]Br treatments, and the morphology of cells
changed gradually from fusiform to round. The SOD and CAT activities
increased in concentrations lower than the IC50, whereas they were
inhibited when the concentration was higher than IC50. There was a lin-
ear regression between ROS level and growth inhibition, cellular mem-
brane permeability, and chlorophyll concentration. The results
indicated that the increase in ROS content in algal cells is the important
toxicological mechanism of [DPy]Br to S. obliquus. The toxicity of pyri-
dine ILs, using [DPy]Br as a sample, should be taken into consideration.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2017.10.021.
Acknowledgements
This work was supported by the National Natural Science Founda-
tion of China (No. 21377115), the Zhejiang Provincial Natural Science
Foundation of China (No. LY18B070002).
References
Amde, M., Liu, J.F., Pang, L., 2015. Environmental application, fate, effects, and concerns of
ionic liquids: a review. Environ. Sci. Technol. 49, 12611–12627.
Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal
transduction. Annu. Rev. Plant Biol. 55 (x), 373–399.
Ashraf, M., Harris, P.J.C., 2013. Photosynthesis under stressful environments: an overview.
Photosynthetica 51 (2), 163–190.
Baker, N.R., 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu. Rev.
Plant Biol. 59, 89–113.
Bernard, V., Aline, P., Michel, L., Canbell, P., 2000. Permeability changes in model and phy-
toplankton membranes in the presence of aquatic humic substances. Environ. Sci.
Technol. 34 (18), 3907–3913.
Bradford, E., 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem.
72, 248–254.
Bubalo, M.C., Radošević, K., Redovniković, I.R., Halambek, J., Srček, V.G., 2014. A brief over-
view of the potential environmental hazards of ionic liquids. Ecotoxicol. Environ. Saf.
99 (1), 1–2.
Calabrese, E.J., 2015. Hormesis: principles and applications. Homeopathy 104 (2), 69–82.
1580 D. Liu et al. / Science of the Total Environment 622–623 (2018) 1572–1580
CNEPA (Chinese National Environmental Protection Agency), 1990. Algal Growth
Inhibiting Test. Guidelines for Testing of Chemicals. The Chinese Chemical Industry
Press, Beijing, China.
Corcoll, N., Ricart, M., Franz, S., Sanspiché, F., Schmittjansen, M., Guasch, H., 2012. The use
of photosynthetic fluorescence parameters from autotrophic biofilms for monitoring
the effect of chemicals in river ecosystems. Emerging and Priority Pollutants in Rivers.
32, pp. 85–116.
Cvjetko, B.M., Radoševié, K., Radojčić, I.R., Halambek, J., Srček, V.G., 2014. A brief overview
of the potential environmental hazards of ionic liquids. Ecotoxicol. Environ. Saf. 99,
1–12.
Deng, X.Y., Chen, B., Li, D., Hu, X.L., Cheng, J., Gao, K., Wang, C.H., 2017a. Growth and phys-
iological responses of a marine diatom (Phaeodactylum tricornutum) against two
imidazolium-based ionic liquids ([C4mim]BF4 and [C8mim]BF4). Aquat. Toxicol. 189,
115–122.
Deng, X.Y., Li, D., Wang, L., Hu, X.L., Cheng, J., Gao, K., 2017b. Potential toxicity of ionic liq-
uid ([C12mim]BF4) on the growth and biochemical characteristics of a marine diatom
Phaeodactylum tricornutum. Sci. Total Environ. 586, 675–684.
Egorova, K.S., Ananikov, V.P., 2014. Toxicity of ionic liquids: eco(cyto)activity as compli-
cated, but unavoidable parameter for task-specific optimization. ChemSusChem 7,
336–360.
Egorova, K.S., Gordeev, E.G., Ananikov, V.P., 2017. Biological activity of ionic liquids and
their application in pharmaceutics and medicine [J]. Chem. Rev. 117, 7132–7189.
Feierabend, J., Engel, S., 1986. Photoinactivation of catalase in vitro and in leaves. Arch.
Biochem. Biophys. 251 (2), 567–576.
Fu, L., Li, J.J., Wang, Y., Wang, X.H., Wen, Y., Qin, W.C., Su, M.L., Zhao, Y.H., 2015. Evaluation
of toxicity data to green algae and relationship with hydrophobicity. Chemosphere
120, 16–22.
Gal, N., Malferarri, D., Kolusheva, S., Galletti, P., Tagliavini, E., Jelinek, R., 2012. Membrane
interactions of ionic liquids: possible determinants for biological activity and toxicity.
Biochim. Biophys. Acta 1818 (12), 2967–2974.
Hallett, J.P., Welton, T., 2011. Room-temperature ionic liquids: solvents for synthesis and
catalysis. ChemInform 42 (36), 3508–3576.
Hong, Y., Hu, H.Y., Xie, X., Sakoda, A., Sagehashi, M., Li, F.M., 2009. Gramine-induced
growth inhibition, oxidative damage and antioxidant responses in freshwater cyano-
bacterium Microcystis aeruginosa. Aquat. Toxicol. 91, 262–269.
Kárászová, M., Kacirková, M., Friess, K., Izák, P., 2014. Progress in separation of gases by
permeation and liquids by pervaporation using ionic liquids: a review. Sep. Purif.
Technol. 132, 93–101.
Khare, T., Kumar, V., Kishor, P.B.K., 2015. Na+ and Cl− ions show additive effects under
NaCl stress on induction of oxidative stress and the responsive antioxidative defense
in rice. Protoplasma 252 (4), 1149–1165.
Kim, T.S., Laviale, M., Feurtet-Mazel, A., Jan, G., Gonzalez, P., Mazzella, N., Morin, S., 2015.
Herbicide toxicity on river biofilms assessed by pulse amplitude modulated (PAM)
fluorometry. Aquat. Toxicol. 165, 160–171.
Koppel, D.J., Gissi, F., Adams, M.S., King, C.K., Jolley, D.F., 2017. Chronic toxicity of five
metals to the polar marine microalga Cryothecomonas armigera - application of a
new bioassay. Environ. Pollut. 228, 211–221.
Kumar, K.S., Dahms, H.U., Lee, J.S., Kim, H.C., Lee, W.C., Shin, K.H., 2014. Algal photosyn-
thetic responses to toxic metals and herbicides assessed by chlorophyll a fluores-
cence. Ecotoxicol. Environ. Saf. 104 (2), 51–71.
Laguerre, C., Sanchez-Hernandez, J.C., Köhler, H.R., Triebskorn, R., Capowiez, Y., Rault, M.,
Mazzia, C., 2009. B-type esterases in the snail Xeropicta derbentina: an enzymological
analysis to evaluate their use as biomarkers of pesticide exposure. Environ. Pollut.
157 (1), 199–207.
Lefebvre, S., Mouget, J.L., Lavaud, J., 2011. Duration of rapid light curves for determining
the photosynthetic activity of microphytobenthos biofilm in situ. Aquat. Bot. 95 (1),
1–8.
Lichtenthaler, H.K., Buschmann, C., Knapp, M., 2005. How to correctly determine the dif-
ferent chlorophyll fluorescence parameters and the chlorophyll fluorescence de-
crease ratio RFd of leaves with the PAM fluorometer. Photosynthetica 43 (3),
379–393.
Liu, H.J., Zhang, X.Q., Chen, C., Du, S.T., Dong, Y., 2015a. Effects of imidazolium chloride
ionic liquids and their toxicity to Scenedesmus obliquus. Ecotoxicol. Environ. Saf.
122, 83–90.
Liu, H.J., Zhang, S.X., Zhang, X.Q., Chen, C.D., 2015b. Growth inhibition and effect on pho-
tosystem by three imidazolium chloride ionic liquids in rice seedlings. J. Hazard.
Mater. 286, 440–448.
Liu, H.J., Xia, Y., Cai, W., Zhang, Y., Zhang, X., Du, S., 2017. Enantioselective oxidative stress
and oxidative damage caused by Rac- and S-metolachlor to Scenedesmus obliquus.
Chemosphere 173, 22–30.
Luo, X.Y., Guo, Y., Ding, F., Zhao, H.Q., Cui, G.K., Li, H.R., Wang, C.M., 2014. Significant im-
provements in CO2 capture by pyridine-containing anion-functionalized ionic liquids
through multiple-site cooperative interactions. Angew. Chem. 53 (27), 7053–7507.
Mackinney, G., 1941. Absorption of light by chlorophyll solutions. Biol. Chem. 140 (2),
315–322.
Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence - a practical guide. J. Exp. Bot.
51, 659–668.
Nancharaiah, Y.V., Francis, A.J., 2015. Hormetic effect of ionic liquid 1-ethyl-3-
methylimidazolium acetate on bacteria. Chemosphere 128, 178–183.
Organization for Economic Co-operation and Development (OECD), 2006. Guideline for
testing of chemicals: algal growth inhibition test. OECD Guideline, p. 201.
Pacurari, M., Qian, Y., Fu, W., Schwegler-Berry, D., Ding, M., Castranova, V., Guo, N.L., 2012.
Cell permeability, migration, and reactive oxygen species induced by multiwalled
carbon nanotubes in human microvascular endothelial cells. J. Toxic. Environ. Health
A 75 (2), 112–128.
Petrová, Š., Rezek, J., Soudek, P., Vaněk, T., 2017. Preliminary study of phytoremediation of
brownfield soil contaminated by PAHs. Sci. Total Environ. 599-600, 572–580.
Qian, H.F., Zhu, K., Lu, H.P., Lavoie, M., Chen, S., Zhou, Z., Deng, Z., Chen, J., Fu, Z., 2016. Con-
trasting silver nanoparticle toxicity and detoxification strategies in Microcystis
aeruginosa and Chlorella vulgaris: new insights from proteomic and physiological
analyses. Sci. Total Environ. 572, 1213–1221.
Rantamäki, A., Ruokonen, S., Sklavounos, E., Kyllönen, L., King, A., Wiedmer, S., 2017. Im-
pact of surface-active guanidinium-, tetramethylguanidinium-, and cholinium-based
ionic liquids on Vibrio fischeri cells and Dipalmitoylphosphatidylcholine liposomes. Sci
Rep 7, 46673.
Saleh, N.B., Milliron, D.J., Aich, N., Katz, L.E., Liljestrand, H.M., Kirisits, M.J., 2016. Impor-
tance of doping, dopant distribution, and defects on electronic band structure alter-
ation of metal oxide nanoparticles: implications for reactive oxygen species. Sci.
Total Environ. 568, 926–932.
Schreiber, U., Quayle, P., Schmidt, S., Escher, B., Mueller, J., 2007. Methodology and evalu-
ation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluores-
cence imaging. Biosens. Bioelectron. 22 (11), 2554–2563.
Stefanie, K., Katja, K., 2008. The role of reactive oxygen species in copper toxicity to two
freshwater green algae. J. Phycol. 44 (2), 311–319.
Wang, J., Zhu, J.Y., Liu, S.P., Liu, B.Y., Gao, Y.N., Wu, Z.B., 2011. Generation of reactive oxy-
gen species in cyanobacteria and green algae induced by allelochemicals of sub-
merged macrophytes. Chemosphere 85, 977–982.
Wen, Y.Z., Chen, H., Shen, C., Zhao, M., Liu, W., 2011. Enantioselectivity tuning of chiral
herbicide dichlorprop by copper: roles of reactive oxygen species. Environ. Sci.
Technol. 45 (11), 4778–4784.
Zhang, C., Zhu, L.S., Wang, J.H., Wang, J., Zhou, T.T., Xu, Y.Q., Cheng, C., 2017. The acute
toxic effects of imidazolium-based ionic liquids with different alkyl-chain lengths
and anions on zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 140, 235–240.
Zhu, C.J., Peng, Y., Tong, Z.H., Lu, L.Y., Cui, Y.H., Yu, H.Q., 2016. Hormetic effect and mech-
anism of imidazolium-based ionic liquids on the nematode Caenorhabditis elegans.
Chemosphere 157, 65–70.
Abbreviations
ILs: ionic liquids
[DPy]Br: 1-decylpyridinium bromide
FDA: fluorescein acetate
Chl a: chlorophyll a
Chl b: chlorophyll b
Chl(a + b): chlorophyll (a + b)
IC50: half inhibitory concentration
F0: the minimum fluorescence
Fm: maximum fluorescence
Fv: the variable fluorescence
Fv/Fm: the maximum effective quantum yield of PSII (in the dark-adapted state)
Fv/F0: maximum quantum yield of PSII photochemistry
Y(II): yield of photochemical quantum
NPQ: non-photochemical quenching coefficient
PAR: photosynthetically active radiation
ROS: reactive oxygen species
SOD: superoxide dismutase
CAT: catalase